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Primer group and probe for detecting human hepatitis B virus

A hepatitis B virus, primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc.

Active Publication Date: 2014-06-11
QIAGEN SHENZHEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ELISA method has always been a traditional method for clinical diagnosis of HBV infection. It reflects the immune status of the body’s HBV infection by detecting serum immunological markers; this method is simple and easy to implement, and has been successfully used in the diagnosis of hepatitis B virus infection, but the sensitivity of this method is not high , cannot be quantified
The detection of HBV-DNA uses polymerase chain reaction (PCR) technology to specifically amplify the gene fragments in the conserved region of the hepatitis B virus genome, and with an index close to 2n (n is the number of cycles), a very small amount of HBV-DNA specific molecular fragment amplified to 1x10 7 ~1x10 8 It can detect a very small amount of nucleic acid in the specimen, which can be detected a few days after the virus infection, which can greatly shorten the "window period", greatly improve the detection rate of HBV, and provide a basis for clinical diagnosis of HBV infection However, conventional polymerase chain reaction (PCR) technology is only a qualitative detection, which can detect the presence or absence of hepatitis B virus, but cannot determine the viral load in the body, which is not conducive to the evaluation of the treatment effect of hepatitis B

Method used

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  • Primer group and probe for detecting human hepatitis B virus
  • Primer group and probe for detecting human hepatitis B virus
  • Primer group and probe for detecting human hepatitis B virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Material

[0097] 1. Kit

[0098] 1.1 The kit of the present invention includes the following components: HBV HS PCR solution, Hot star Taq plus polymerase (5 U / μl) and UNG (1 U / μl)).

[0099] HBV HS PCR solution contains the following components: ultrapure water, Multiplex Virus PCR buffer 20x, Q-solution 5x, 25mmol / L MgCl 2 , 100mmol / L dATP, 100mmol / L dCTP, 100mmol / L dGTP, 100mmol / L dUTP, primer set 1 (forward primer 1 and reverse primer 1), probe 1 (probe 1), primer set 2 (forward Toward Primer 2 and Reverse Primer 2) and Probe 2 (Probe 2).

[0100] 1.2 Control Kit

[0101]The control kit was "Hepatitis B virus (HBV) nucleic acid quantitative detection kit (PCR-fluorescent probe method)" produced by Kaijie Bioengineering (Shenzhen) Co., Ltd. (trade name: careHBV PCR ASSAY; approval number: National Food and Drug Administration (quasi) word 2009 No. 3401037).

[0102] 1.3 Third-party kits

[0103] The third-party kit is COBAS AmpliPrep / COBAS TaqMan HBV Test, v...

Embodiment 2

[0162] Embodiment 2 — the lowest limit of detection (LOD) of kit of the present invention

[0163] Using the WHO quantitative standard described in Example 1, dilute to 200, 100, 30, 20, 10 IU / ml with negative plasma (IU / ml represents the amount of virus contained per milliliter). Standard HBV DNA was prepared by the method described in Example 1. Using the kit of the present invention, each concentration gradient was repeatedly detected 25 times through fluorescent quantitative PCR reaction. Determine the detection rate of the kit of the present invention at each standard concentration, thereby determining the minimum detection limit. The result is as figure 1 As shown, the detection rate of 20 IU / ml samples can reach 96%.

Embodiment 3

[0164] Embodiment 3—the quantitative detection limit (LOQ) of kit of the present invention

[0165]Using the WHO quantitative standard, L0 standard and B / C / D genotype standard described in Example 1, dilute to 40 and 20 IU / ml with negative plasma, respectively. The HBV DNA of the standard was prepared by the method described in Example 1. Using 3 batches of the kit of the present invention, each concentration gradient was repeatedly detected 25 times by fluorescent quantitative PCR reaction.

[0166] Determine the quantitative detection limit (LOQ) of the kit of the present invention according to the following criteria: calculate the logarithmic value of the quantitative result, count the ratio (quantitative accuracy rate) of the logarithmic value of the quantitative result under each sample concentration within the range of ± 0.5 log10 of the theoretical value, The ratio should be greater than or equal to 22 / 25.

[0167] Experimental results such as figure 2 As shown, w...

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Abstract

The invention relates to a primer group and / or a probe for specific detection of human hepatitis B virus (HBV). The invention aslo relates to a kit and a microarray which comprise the primer groups and / or the probes and are used for specific detection of the human hepatitis B virus (HBV). The invention aslo relates to applications of the primer group and / or the probe in preparation of the kit and the microarray for specific detection of the hepatitis B virus in a sample. The invention aslo relates to a method of for detecting HBV genes in the sample by using the primer group and / or the probe.

Description

technical field [0001] The present invention relates to primer sets and / or probes for the specific detection of human hepatitis B virus (HBV). The present invention also relates to kits and microarrays for specific detection of human hepatitis B virus comprising primer sets and / or probes. The present invention also relates to the use of primer sets and / or probes in the preparation of kits and microarrays for specific detection of hepatitis B virus in samples. The present invention also relates to a method for detecting HBV genes in samples using primer sets and / or probes. Background technique [0002] The 5-year mortality rates of chronic hepatitis B, compensated and decompensated cirrhosis are 0%-2%, 14%-20% and 70%-86%, respectively, and the related liver diseases caused by chronic hepatitis B are HBV infection is the main cause of death. In China, more than 300,000 deaths from liver cirrhosis and liver cancer are caused by HBV infection every year. Therefore, active in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/70C12Q2531/113C12Q2563/107
Inventor 陈华彭进
Owner QIAGEN SHENZHEN CO LTD
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